P-type ATPases are cell-membrane pumps that are of fundamental importance in eukaryotes. The ion gradients created by Ca2+-ATPases or Na+,K+-ATPases are involved in signalling, maintaining the membrane potential as well as secondary active transport. Despite the recent progress in membrane protein crystallography, the lack of detailed structural information currently prevents a better understanding of function and specificity of cation pumps. One specific aim of this proposed research is to determine the structure of the plasma-membrane Ca2+-ATPase (PMCA) and to understand its regulatory mechanism. The autoinhibited PMCA will be recombinantly expressed in yeast, purified and crystallized. In addition, it is also planned to determine the structure of the activated complex consisting of PMCA and calcium-bound calmodulin (Ca2+-CaM). In order to deepen our understanding of cation pumps in their native environment, I further propose to investigate Na+,K+-ATPases in native membranes using a multi-technique approach. First, I will analyze complexes of Na+,K+-ATPase isolated from native membranes by mass spectrometry to identify peripheral proteins. In the next step, I plan to assemble those identified multi-protein complexes in vitro and structurally characterize them by cryo-EM. Finally, I plan to use AFM to investigate the oligomeric arrangement of Na+,K+-ATPases in native membranes. This multi-disciplinary project will hopefully expand our knowledge of cation pumps by putting atomic details in the context of the native environment and will concurrently allow me to gain experience in membrane protein crystallography that will complement my expertise in biophysical methods.
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